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Abstract:

The invention concerns promoters, in particular for the expression of
genes and/or coding sequences in vaccinia viruses such as Modified
vaccinia virus Ankara (MVA). The invention further concerns expression
cassettes comprising said promoter, vectors comprising said expression
cassettes as well as pharmaceutical compositions and vaccines.

Claims:

1-22. (canceled)

23. An isolated DNA consisting of SEQ ID NO:2, wherein the isolated DNA is
active as a modified vaccinia virus Ankara promoter or active as a
promoter in vaccinia virus infected cells.

24. An isolated DNA consisting of a derivative of SEQ ID NO:2;wherein no
more than 10 nucleotides are substituted in the derivative relative to
the sequence of SEQ ID NO:2; andwherein the isolated DNA is active as a
modified vaccinia virus Ankara promoter or active as a promoter in
vaccinia virus infected cells.

25. The isolated DNA of claim 24, wherein no more than 5 nucleotides are
substituted in the derivative relative to the sequence of SEQ ID NO:2.

26. An expression cassette comprising:a promoter comprising SEQ ID NO:2;
anda coding sequence operably linked to the promoter;wherein the
expression of the coding sequence is controlled by the promoter;
andwherein the expression cassette is not an expression cassette that
naturally occurs in the genome of a vaccinia virus.

27. An expression cassette comprising:a promoter comprising a derivative
of SEQ ID NO:2,wherein no more than 10 nucleotides are substituted in the
derivative relative to the sequence of SEQ ID NO:2, anda coding sequence
operably linked to the promoter,wherein the expression of the coding
sequence is controlled by the promoter, andwherein the expression
cassette is not an expression cassette that naturally occurs in the
genome of a vaccinia virus.

28. The expression cassette of claim 27, wherein no more than 5
nucleotides are substituted in the derivative relative to the sequence of
SEQ ID NO:2.

34. The vector of claim 33, wherein the vaccinia virus is strain MVA-BN
deposited at the European Collection of Cell Cultures (ECACC) under
number V00083008, strain MVA 572 deposited at ECACC under number
V94012707, or strain MVA 575 deposited under number V00120707 at ECACC.

35. A host cell comprising the vector of claim 30.

36. A composition for inducing an immunological response in a living
animal body comprising the viral vector of claim 31.

37. A method for introducing a coding sequence into a target cell
comprising introducing the vector of claim 30 into the target cell.

38. A method for producing a peptide, protein, or virus comprising
infection of a host cell with the viral vector of claim 31 and
cultivating the infected host cell under suitable conditions.

39. The vector of claim 33, wherein the expression cassette is inserted
into a naturally occurring deletion site of the MVA genome.

40. The vector of claim 33, wherein the expression cassette is inserted
into an intergenic region of the MVA genome.

47. The vector of claim 46, wherein the vaccinia virus is strain MVA-BN
deposited at the European Collection of Cell Cultures (ECACC) under
number V00083008, strain MVA 572 deposited at ECACC under number
V94012707, or strain MVA 575 deposited under number V00120707 at ECACC.

48. A host cell comprising the vector of claim 43.

49. A composition for inducing an immunological response in a living
animal body comprising the viral vector of claim 44.

50. A method for introducing a coding sequence into a target cell
comprising introducing the vector of claim 43 into the target cell.

51. A method for producing a peptide, protein, or virus comprising
infection of a host cell with the viral vector of claim 44 and
cultivating the infected host cell under suitable conditions.

52. The vector of claim 46, wherein the expression cassette is inserted
into a naturally occurring deletion site of the MVA genome.

53. The vector of claim 46, wherein the expression cassette is inserted
into an intergenic region of the MVA genome.

60. The vector of claim 59, wherein the vaccinia virus is strain MVA-BN
deposited at the European Collection of Cell Cultures (ECACC) under
number V00083008, strain MVA 572 deposited at ECACC under number
V94012707, or strain MVA 575 deposited under number V00120707 at ECACC.

61. A host cell comprising the vector of claim 56.

62. A composition for inducing an immunological response in a living
animal body comprising the viral vector of claim 57.

63. A method for introducing a coding sequence into a target cell
comprising introducing the vector of claim 56 into the target cell.

64. A method for producing a peptide, protein, or virus comprising
infection of a host cell with the viral vector of claim 57 and
cultivating the infected host cell under suitable conditions.

65. The vector of claim 59, wherein the expression cassette is inserted
into a naturally occurring deletion site of the MVA genome.

66. The vector of claim 59, wherein the expression cassette is inserted
into an intergenic region of the MVA genome.

67. A composition comprising the vector of claim 56 with an adjuvant.

Description:

[0001]The invention concerns promoters, in particular for the expression
of genes and/or coding sequences in vaccinia viruses such as Modified
vaccinia virus Ankara (MVA). The invention further concerns expression
cassettes comprising said promoter, vectors comprising said expression
cassettes as well as pharmaceutical compositions and vaccines.

BACKGROUND OF THE INVENTION

[0002]Recombinant poxviruses are widely used to express foreign antigens
in infected cells. Moreover, recombinant poxviruses are currently tested
as very promising vaccines to induce an immune response against the
foreign antigen expressed from the poxvirus vector. Most popular are
avipoxviruses on the one side and vaccinia viruses, in particular
Modified vaccinia virus Ankara (MVA) on the other side. MVA is related to
vaccinia virus, a member of the genera Orthopoxvirus in the family of
Poxyiridae. MVA has been generated by 516 serial passages on chicken
embryo fibroblasts of the Ankara strain of vaccinia virus (CVA) (for
review see Mayr, A., et al. Infection 3, 6-14 [1975]). As a consequence
of these long-term passages the resulting MVA virus deleted about 31
kilobases of its genomic sequence and, therefore, was described as highly
host cell restricted to avian cells (Meyer, H. et al., J. Gen. Virol. 72,
1031-1038 [1991]). It was shown, in a variety of animal models that the
resulting MVA was significantly aviruient (Mayr, A. & Danner, K. [1978]
Dev. Biol. Stand. 41: 225-34). Additionally, this MVA strain has been
tested in clinical trials as vaccine to immunize against the human
smallpox disease (Mayr et al., Zbl. Bakt. Hyg. I, Abt. Org. B 167,
375-390 [1987], Stickl et al., Dtsch. med. Wschr. 99, 2386-2392 [1974]).

[0004]For the expression of heterologous genes in pox viruses only a few
promoters are known to the person skilled in the art, such as the 30K and
40K promoters (see e.g. U.S. Pat. No. 5,747,324), a strong synthetic
early/late promoter (see e.g. Sutter et al., Vaccine (1994) 12, 1032-40),
the P7.5 promoter (see e.g. Endo et al., J. Gen. Virol. (1991) 72,
699-703) and the promoter derived from the cowpox virus A-type inclusion
(ATI) gene (Li et al., J. Gen. Virol. (1998) 79, 613). All of these
promoters have been used in recombinant vaccinia viruses to express
heterologous genes and were shown to express said genes resulting in the
production of the protein encoded by the heterologous gene. Since only a
few promoters are available for the expression of genes in vaccinia virus
expression systems there is general need for alternative promoters in
vaccinia viruses. In addition, all of the promoters known so far are
rather strong late promoters, i.e. useful for the expression of genes
after the replication of the vaccinia virus vector has occurred. For some
application it is desirable to have promoters allowing the expression of
genes immediately after the infection of the cells, i.e. there is a need
for vaccinia virus early promoters.

[0005]Moreover, as pointed out above, MVA is a very promising virus for
the expression of heterologous genes due to its improved safety profile.
However, all promoters known so far for the expression of heterologous
genes in MVA were derived from other vaccinia viruses or are synthetic
promoters for the expression in other vaccinia virus. Thus, there is also
a need for promoters optimized for the expression in MVA.

DETAILED DESCRIPTION OF THE INVENTION

[0006]The invention concerns promoters derived from the genome of Modified
vaccinia virus Ankara (MVA). MVA promoters were not yet known in the art.

[0007]In particular the invention concerns a promoter comprising or
consisting of a nucleotide sequence selected from the group comprising:

(i) the nucleotide sequence of anyone of the following SEQ ID NO: 1 to 6:

(ii) subsequences of the sequence according to anyone of SEQ ID: No. 1 to
6(iii) sequences having one or more nucleotide substitutions, deletions
and/or insertions with respect to the sequences as defined in (i) or
(ii).

[0008]SEQ ID NO: 1 to 6 are naturally part of the MVA genome and are
located upstream of the reading frames A42R, J6R, F6R, I2R, C7L and B9R,
respectively.

[0009]The promoters according to the present invention are preferably
active as vaccinia virus promoters or active as promoters in vaccinia
virus infected cells. The vaccinia virus is preferably MVA, in particular
one of the MVA strains as specified below. "Active as vaccinia virus
promoter" means that the promoter is able to direct the expression of a
gene to which it is operably linked in a vaccinia virus after infection
of cells with said virus. The cells are preferably cells that allow late
and/or early expression of the vaccinia virus. "A promoter active in
vaccinia virus infected cells" includes also the situation in which the
promoter is not part of a vaccinia virus genome, e.g. part of a plasmid
or a non-vaccinia virus viral genome; in such a situation the promoter
according to the present invention is active if the cell comprising the
promoter also comprises a vaccinia virus genome, e.g. if the cell is
infected with a vaccinia virus. Under these circumstances the viral RNA
polymerase recognizes the promoter according to the present invention and
the expression of the gene/coding sequence that is linked to the promoter
is activated.

[0010]According to the present invention it is possible to use anyone of
the promoters as specified in SEQ ID NO: 1 to SEQ ID NO: 6. The promoter
that is actually used to direct the expression of the gene/coding
sequence may consist of anyone of SEQ ID NO: 1 to SEQ ID NO: 6 or the
actually used promoter may be a larger structure that comprises anyone of
SEQ ID NO: 1 to SEQ ID NO: 6. Alternatively it is within the scope of the
present invention to use a derivative of these promoters, which may be a
subsequence of the sequences as defined in anyone of SEQ. ID NO: 1 to 6.
The term "subsequence of the sequences according to anyone of SEQ ID NO:
1 to 6" refers to shorter fragments of anyone of SEQ ID NO: 1 to 6 that
are still active as a promoter, in particular as promoter in vaccinia
virus or in vaccinia virus infected cells. Again, the vaccinia virus is
preferably MVA, such as one of the strains to specified below. A typical
subsequence of anyone of SEQ ID NO:1 to SEQ ID NO: 6 has a length of at
least 15 nucleotides, more preferably of at least 20 nucleotides, even
more preferably of at least 25 nucleotides, most preferably of at least
30 nucleotides of anyone of the sequences of SEQ ID NO:1 to SEQ ID NO: 6.

[0012]The derivative of the promoter comprising or consisting of a
nucleotide sequence of anyone of SEQ ID NO: 1 to 6 or subsequences
thereof, in particular the derivative of the nucleotide sequence of SEQ
ID NO: 7 to SEQ ID NO: 12 can also be a sequence that has one or more
nucleotide substitutions, deletions and/or insertions with respect to any
one of the sequences of SEQ ID NO:1 to 6 or subsequences thereof, in
particular of the nucleotide sequences of SEQ ID NO: 7 to SEQ ID NO: 12.
The derivatives according to the present invention are still active as a
promoter, in particular as vaccinia virus promoter in a vaccinia virus or
in vaccinia virus infected cells, more preferably as MVA promoter in MVA
or in MVA infected cells. A sequence having one or more nucleotide
substitutions is a sequence in which one or more nucleotides of the
sequence according to anyone of SEQ ID NO: 1 to 6 or subsequences
thereof, such as the sequences according to anyone of SEQ ID NO: 7 to 12
are substituted by different nucleotides. A sequence having one or more
nucleotide insertions is a sequence in which one or more nucleotides are
inserted at one or more locations anyone of SEQ ID NO: 1 to 6 or
subsequences thereof, in particular of the nucleotide sequences of SEQ ID
NO: 7 to SEQ ID NO: 12. A sequence having one or more nucleotide
deletions is a sequence in which one or more nucleotides of the sequence
according anyone of SEQ ID NO: 1 to 6 or subsequences thereof, in
particular of the nucleotide sequences of SEQ ID NO: 7 to SEQ ID NO: 12
are deleted. In the derivatives according to the present invention
deletions, substitutions and insertions may be combined in one sequence.

[0013]An example of an derivative of a subsequence according to the
present invention is SEQ ID NO: 10, which is a subsequence of SEQ ID NO:
3 having in addition one nucleotide exchange with respect to the
corresponding nucleotide sequence in SEQ ID NO: 3.

[0014]Preferably the derivative has a homology of at least 40%, more
preferably of at least 60%, even more preferably of at least 80%, most
preferably of at least 90% when compared to anyone of the sequence of SEQ
ID NO: 1 to SEQ ID NO: 6 or subsequences thereof, in particular of the
sequences according to anyone of SEQ ID NO: 7 to SEQ ID NO: 12. According
to the most preferred embodiment not more than 10 nucleotides, even more
preferably not more than 5 nucleotides are substituted, deleted and/or
inserted in the sequence of anyone of SEQ ID NO: 7 to SEQ ID NO: 12.

[0015]A bundle of prior art documents allows the person skilled in the art
to predict which derivatives or subsequences of anyone of SEQ ID NO: 1 to
12 still have the biological activity of being active as a vaccinia virus
promoter, in particular as a promoter active in MVA. In this context
reference is made to Chakrarbarti et al., Biotechniques (1997) 23,
1094-1097 and Davison and Moss, J. Mol. Biol. (1989) 210, 771-784.
Moreover, whether a fragment is still active as a vaccinia virus
promoter, in particular as a MVA promoter can easily be evaluated by a
person skilled in the art. In particular the sequence derivative can be
cloned upstream of a reporter gene in a plasmid construct. Said construct
may be transfected into a eukaryotic cell or cell line, such as CEF or
BHK cells that has been infected with MVA. The expression of the reporter
gene is determined and compared to the expression of the reporter gene
controlled by the promoter according to anyone of SEQ ID NO: 1 to 6. A
derivative according to the present invention is a derivative having a
promoter activity in said test system of at least 10%, preferably of at
least 30%, more preferably of at least 50%, even more preferably of at
least 70%, most preferably of at least 90% compared to the activity of
the promoter sequence of anyone of SEQ ID NO: 1 to 6. Also those
derivatives of anyone of SEQ ID NO: 1 to 12 are within the scope of the
present invention that have a higher promoter activity.

[0016]The promoters according to the present invention are particularly
suitable for the expression of coding sequences in MVA.

[0017]The promoter according to SEQ ID NO: 1 has a very strong activity,
in particular as late promoter although it can also be used as early
promoter. The same considerations apply for the corresponding
subsequences such as the sequence of SEQ ID NO: 7, which is, however,
particularly useful as late promoter.

[0018]The promoter according to SEQ ID NO: 2 also has a rather strong
activity, in particular as late promoter. It can also be used as early
promoter. The same considerations apply for the corresponding
subsequences, such as the sequence of SEQ ID NO: 8, which is, however,
particularly useful as late promoter.

[0019]The promoter according to SEQ ID NO: 3 is particularly useful as
early promoter and has the highest early promoter activity of all
promoters tested. However, it can also be used as late promoters. The
same considerations apply for the corresponding subsequences, such as the
sequences of SEQ ID NO: 9 and 10, respectively. Of these subsequences SEQ
ID NO: 9 is particularly useful as early promoter and SEQ ID NO: 10 is
particularly useful as late promoter.

[0020]The promoter according to SEQ ID NO: 4 is particularly useful if it
is intended to express a linked coding sequence early and late since this
promoter has a rather high activity under early as well as late
conditions. The same considerations apply for the corresponding
subsequences, such as the sequences of SEQ ID NO: 11 and 12,
respectively. Of these subsequences SEQ ID NO: 11 is particularly useful
as early promoter and SEQ ID NO: 12 is particularly useful as late
promoter.

[0021]The promoters according to SEQ ID NO: 5 and 6 are particularly
useful if it is intended to express linked coding sequences in rather low
amounts. This is sometimes desirably if the linked coding sequence
encodes a toxic gene product and/or if it is intended to induce a high
avidity immune response.

[0022]The term "early promoter" refers to promoters that are active in
vaccinia virus or vaccinia virus infected cells, before viral DNA
replication has occurred. Methods are known to the person skilled in the
art how it can be checked whether a promoter is an early promoter. In
particular, the promoter of interest can be inserted upstream of a
reporter gene. The construct comprising the promoter and the reporter
gene are introduced into cells that are infected with a vaccinia virus.
In order to assess for the activity as early promoter the cells are
incubated with a substance that inhibits the DNA replication such as
AraC. DNA replication is a prerequisite for the late promoter activity.
Thus, any promoter activity that is measured in this assay system is due
to elements active as early promoter. Consequently, the term "late
promoter" refers to any promoters that are active after DNA replication
has taken place. The late activity can also be measured by methods known
to the person skilled in the art. For the sake of simplicity the term
"late promoter" as used in the present application refers to the activity
of a promoter that is determined if no substance is added that blocks DNA
replication.

[0023]According to a further embodiment the present invention refers to an
expression cassette comprising the promoter according to the present
invention and a coding sequence, wherein the expression of the coding
sequence is controlled by said promoter. The expression cassette is
preferably not an expression cassette that occurs naturally in the genome
of a vaccinia virus. Thus, if the promoter according to the present
invention is a promoter that naturally occurs in the genome of the
vaccinia virus the sequence to which the promoter is linked is preferably
different from the sequence to which the promoter is naturally linked in
the vaccinia virus genome. In other words, if the promoter according to
the present invention is identical to a naturally occurring promoter the
coding sequence the expression of which is controlled by the promoter
and/or the sequences located between the promoter and the coding sequence
are different from the corresponding sequences to which said promoter is
naturally linked. The term "different" in this context refers to
sequences that show at least one nucleotide difference in said sequence.
Preferably it is the coding sequence which has at least one nucleotide
difference. According to other alternatives the homology between the
coding sequence in the expression cassette and the sequence to which the
promoter is naturally linked is less than 90%, less than 80%, less than
70%, less than 60%, less than 50%, less than 40% or even less than 20%.
Most preferably the coding sequence that is controlled by the promoter
according to the present invention codes for a peptide/protein having a
difference of at least one amino acid compared to the naturally occurring
protein encoded by said coding sequence. By way of example the expression
cassette is not the expression cassette comprising the naturally
occurring vaccinia virus C7L promoter directing the expression of the
naturally occurring C7L gene, e.g. the expression cassette is not the
expression cassette disclosed in WO2004/015118 comprising the C7L
promoter and the C7L coding sequence.

[0024]On the other hand, if the sequence which should be expressed is a
naturally occurring vaccinia virus sequence the promoter that is used to
express said sequence is different from the promoter that is directs the
expression of the coding sequence in the natural context. According to
this alternative the nucleotide sequence of the promoter differs in at
least one nucleotide from the sequence of the naturally occurring
vaccinia virus promoter. According to other alternatives the homology
between the promoter according to the present invention that controls the
expression of the vaccinia virus sequence and the naturally occurring
promoter linked to the vaccinia virus sequence is less than 90%, less
than 80%, less than 70%, less than 60%, less than 50% or even less than
40%.

[0025]Preferably the coding sequence may code for at least one antigenic
epitope or antigen, therapeutic peptides or proteins, antisense RNA or
ribozymes. If the coding sequence encodes an antigenic epitope or antigen
the expression cassette may be used to express said antigen after
introduction of said expression cassette in cells in an organism, e.g. a
mammalian animal including a human. The presentation of said
antigen/epitope may elicit an immune response in the organism that may
lead to a vaccination of the organism against the agent from which the
antigen/epitope is derived. More specifically the epitope/antigen may be
part of a larger amino acid sequence such as a polyepitope, peptide or
protein. Preferably the coding sequence codes for at least one antigenic
epitope or antigen, therapeutic peptides or proteins, antisense RNA or
ribozymes which are not encoded by a vaccinia virus genome.

[0027]Alternatively the coding sequence may encode a therapeutic compound
such as interleukins, interferons, ribozymes or enzymes.

[0028]In more general terms the invention concerns any nucleic acid
sequence comprising the promoter according to the present invention
and/or the expression cassette according to the present invention. The
nucleic acid may be RNA, e.g. if the promoter is part of a retroviral
genome. More preferably the nucleic acid is DNA. The DNA may be any type
of DNA such as linear, circular, single stranded or double stranded DNA.

[0029]According to a further embodiment the promoter and/or expression
cassette according to the present invention may be part of a vector. The
term "vector" refers to any vectors known to the person skilled in the
art. A vector can be a plasmid vector such as pBR322 or a vector of the
pUC series. More preferably the vector is a virus vector. In the context
of the present invention the term "viral vector" or "virus vector" refers
to an infectious virus comprising a viral genome. In this case the DNA of
the present invention is part of the viral genome of the respective viral
vector. The recombinant viral genome is packaged and the obtained
recombinant vectors can be used for the infection of cells and cell
lines, in particular for the infection of living animals including
humans. Typical virus vectors that may be used according to the present
invention are adenoviral vectors, retroviral vectors or vectors on the
basis of the adeno associated virus 2 (AAV2). Most preferred are poxyiral
vectors. The poxvirus may be preferably a canarypox virus, a fowlpoxvirus
or a vaccinia virus.

[0030]More preferred is modified vaccinia virus Ankara (MVA) (Sutter, G.
et al. [1994], Vaccine 12: 1032-40; Antoine, G. et al. [1998], Virology
244: 365-396). The term "MVA" as used in the present application refers
to any MVA strain known in the prior art. An example for an MVA strain is
deposit VR-1508, deposited at the American Type Culture collection
(ATCC), Manassas, Va. 20108, USA. Further examples for MVA virus strains
used according to the present invention are strains MVA 572 and 575
deposited at the European Collection of Animal Cell Cultures (ECACC),
Salisbury (UK) with the deposition number ECACC V94012707 and ECACC
V00120707, respectively, MVA-BN with the deposition number ECACC
V00083008.

[0031]The most preferred MVA-strain is MVA-BN or a derivative thereof. The
features of MVA-BN, the description of biological assays allowing to
evaluate whether a MVA strain is MVA-BN or a derivative thereof and
methods allowing to obtain MVA-BN or a derivative thereof are disclosed
in WO 02/42480. The content of this application is included in the
present application by reference. In particular, reference is made to the
definition of the properties of vaccinia virus according to the invention
as described in WO 02/42480, such as the properties of MVA and the
properties and definitions of the derivates of MVA-BN. Said reference
also discloses how MVA and other vaccinia viruses can be propagated.
Briefly, eukaryotic cells are infected with the virus. The eukaryotic
cells are cells that are susceptible to infection with the respective
poxvirus and allow replication and production of infectious virus. For
MVA an example for this type of cells are chicken embryo fibroblasts
(CEF) and BHK cells (Drexler I., Heller K., Wahren B., Erfle V. and
Sutter G. "Highly attenuated modified vaccinia Ankara replicates in baby
hamster kidney cells, a potential host for virus propagation, but not in
various human transformed and primary cells" J. Gen. Virol. (1998), 79,
347-352). CEF cells can be cultivated under conditions known to the
person skilled in the art. Preferably the CEF cells are cultivated in
serum-free medium in stationary flasks or roller bottles. The incubation
preferably takes place 48 to 96 hours at 37° C.±2° C.
For the infection MVA is preferably used at a multiplicity of infection
(MOI) of 0.05 to 1 TCID50 and the incubation preferably takes place
48 to 72 hours at 37° C.±2° C.

[0032]Methods are known to the person skilled in the art how the
expression cassette or the promoter according to the present invention
can be inserted into a viral genome, in particular into the genome of a
vaccinia virus, most preferably into the genome of MVA. By way of
example, the expression cassette or the promoter or derivative thereof
according to the present invention may be inserted into the genome of MVA
by homologous recombination. To this end a nucleic acid is transfected
into a permissive cell line such as CEF or BHK cells, wherein the nucleic
acid comprises the expression cassette or the promoter or derivative
thereof according to the present invention flanked by nucleotide
stretches that are homologous to the region of the MVA genome in which
the expression cassette or the promoter or derivative thereof according
to the present invention is to be inserted. The cells are infected by MVA
and in the infected cells homologous recombination occurs between the
nucleic acid and the viral genome. Alternatively it is also possible to
first infect the cells with MVA and then to transfect the nucleic acid
into the infected cells. Again recombination occurs in the cells. The
recombinant MVA is then selected by methods known in the prior art. The
construction of recombinant MVA is not restricted to this particular
method. Instead, any suitable method known to the person skilled in the
art may be used to this end.

[0033]The expression cassette or the promoter according to the present
invention may be introduced into any suitable part of the vector, in
particular into a viral, genome. In case of vaccinia viruses the
insertion may be made into non-essential parts of the viral genome or
into an intergenic region of the viral genome. The term "intergenic
region" refers preferably to those parts of the viral genome located
between two adjacent genes that do not comprise coding sequences. If the
vector is MVA the insertion may also be made into a naturally occurring
deletion site of the viral genome. The term "naturally occurring deletion
site" refers to those parts of the viral genome that are deleted with
respect to the genome of the vaccinia virus Copenhagen strain. However,
the insertion sites are not restricted to these preferred insertion sites
in the vaccinia virus genome and the MVA genome, since it is within the
scope of the present invention that the expression cassette may be
inserted anywhere in the viral genome as long as it is possible to obtain
recombinants that can be amplified and propagated in at least one cell
culture system, such as Chicken Embryo Fibroblasts (CEF cells).

[0034]The promoter according to the present invention may be used to
express a gene that is already part of the vector, e.g. the genome of
MVA. Such a gene may be a gene that is naturally part of the viral genome
or a foreign gene that has already been inserted into the vector. In
these cases the promoter according to the present invention is inserted
upstream of the gene in the vector, the expression of which is to be
controlled by the promoter. A MVA vector comprising an expression
cassette according to the present invention can also be made by replacing
anyone of the open reading frames A42R, J6R, F6R, I2R, C7L and B9R by a
coding sequence the expression of which is to be controlled by the
promoter naturally controlling the expression of anyone of said open
reading frames. Thus, by way of example the A42R coding sequence or parts
thereof may be replaced by the coding sequence, which is to be expressed.
In the resulting construct said coding sequence is controlled by a
promoter according to the present invention, namely by the promoter
sequence according to SEQ ID NO: 1 and SEQ ID NO: 7. These expression
cassettes are also within the scope of the present invention.

[0035]According to a further embodiment the invention concerns the vector
according to the present invention as vaccine or medicament. In more
general term the invention relates to a vaccine or pharmaceutical
composition comprising an expression cassette, a DNA or a vector
according to the present invention. Methods are known to the person
skilled in the art how the vaccine or pharmaceutical composition can be
administered to the animal or human body. In case of DNA and plasmid
vectors the DNA and the vector can simply be administered by injection.
If the vector is a viral vector such as a vaccinia virus vector, in
particular a MVA vector it may also be administered to the animal or
human body according to the knowledge of the person skilled in the art,
e.g. by intra venous, intra muscular, intra nasal, intra dermal or
subcutaneous administration. Further details on the amount of virus
administered are given below.

[0037]For the preparation of pharmaceutical compositions or vaccines, the
DNA, expression cassette or vector according to the present invention, in
particular a recombinant vaccinia virus such as recombinant MVA is
converted into a physiologically acceptable form. For vaccinia viruses,
in particular MVA this can be done based on the experience in the
preparation of poxvirus vaccines used for vaccination against smallpox
(as described by Stickl, H. et al. [1974] Dtsch. med. Wschr. 99,
2386-2392). For example, the purified virus is stored at -80° C.
with a titre of 5×108 TCID50/ml formulated in about 10 mM
Tris, 140 mM NaCl pH 7.4. For the preparation of vaccine shots, e.g.,
101-109 particles of the recombinant virus according to the
present invention are lyophilized in phosphate-buffered saline (PBS) in
the presence of 2% peptone and 1% human albumin in an ampoule, preferably
a glass ampoule. Alternatively, the vaccine shots can be produced by
stepwise freeze-drying of the virus in a formulation. This formulation
can contain additional additives such as mannitol, dextran, sugar,
glycine, lactose or polyvinylpyrrolidone or other additives such as
antioxidants or inert gas, stabilizers or recombinant proteins (e.g.
human serum albumin) suitable for in vivo administration. A typical virus
containing formulation suitable for freeze-drying comprises 10 mM
Tris-buffer, 140 mM NaCl, 18.9 g/l Dextran (MW 36000-40000), 45 g/l
Sucrose, 0.108 g/l L-glutamic acid mono potassium salt monohydrate pH
7.4. The glass ampoule is then sealed and can be stored between 4°
C. and room temperature for several months. However, as long as no need
exists the ampoule is stored preferably at temperatures below -20°
C.

[0038]For vaccination or therapy the lyophilisate or the freeze-dried
product can be dissolved in 0.1 to 0.5 ml of an aqueous solution,
preferably water, physiological saline or Tris buffer, and administered
either systemically or locally, i.e. by parenteral, intramuscular or any
other path of administration know to the skilled practitioner. The mode
of administration, the dose and the number of administrations can be
optimized by those skilled in the art in a known manner.

[0039]Thus, according to a related embodiment the invention relates to a
method for affecting, preferably inducing an immunological response in a
living animal body including a human comprising administering the
expression cassette, the DNA, the vector, the pharmaceutical composition
or the vaccine according to the present invention to the animal or human
to be treated. If the vaccine is a vaccinia virus, in particular MVA a
typical vaccine shot for humans comprises at least 102, preferably
at least 104, more preferably at least 106, even more
preferably 107 or 108 TCID50 (tissue culture infectious
dose) of the virus.

[0040]If the vaccine is a recombinant MVA, in particular recombinant
MVA-BN and its derivatives that the virus may be used for prime-boost
administration. Thus, the invention further relates to a method, wherein
the vector is MVA, in particular MVA-BN and its derivatives, and wherein
said vector or the composition or the vaccine comprising said vector is
administered to an animal, including a human in need thereof, in
therapeutically effective amounts in a first inoculation ("priming
inoculation") and in a second inoculation ("boosting inoculation").

[0041]The invention further concerns a method for introducing a coding
sequence into a target cell comprising the introduction of the vector,
the expression cassette or of the DNA according to the present invention
into the target cell. If the vector is a vaccinia virus, in particular
MVA such as MVA-BN the target cell may be a cell in which the virus is
able to replicate such as CEF or BHK cells or a cell that can be infected
by MVA, but in which the virus does not replicate (such as all types of
human cells for MVA-BN).

[0042]The invention further relates to a method for producing a peptide,
protein and/or virus comprising the infection of a host cell with a virus
vector according to the present invention, followed by the cultivation of
the infected host cell under suitable conditions, and further followed by
the isolation and/or enrichment of the peptide and/or protein and/or
viruses produced by said host cell. If it is intended to produce, i.e.
amplify the virus according to the present invention the cell has to be a
cell in which the virus is able to replicate. For vaccinia viruses, in
particular MVA suitable cells are CEF or BHK cells. If it is intended to
produce a peptide/protein encoded by the virus vector according to the
present invention the cell may be any cell that can be infected by the
virus vector and that allows the expression of the virus encoded
proteins/peptides.

[0043]The invention further relates to a method for producing a peptide,
protein and/or virus comprising the transfection of a cell with the
expression cassette, the DNA or the plasmid vector according to the
present invention, followed by the infection of the cell with a vaccinia
virus. The infected host cell is cultivated under suitable conditions. A
further step comprises the isolation and/or enrichment of the peptide
and/or protein and/or viruses produced by said host cell. The step of
infecting the cells with a vaccinia virus may be made before or after the
step of transfection of the cells.

[0044]The invention further relates to cells comprising a promoter, DNA,
expression cassette or vector according to the present invention. In
particular the invention relates to cells infected with the virus vector
according to the present invention.

SHORT DESCRIPTION OF THE FIGURES

[0045]FIG. 1: GUS activity after expression by different promoters

[0046]Cells were infected with MVA-BN and transfected with the appropriate
plasmids. After 48 hours the cells were extracted and the GUS activity
was determined indirectly by measuring the extinction at 415 nm after an
enzymatic reaction, which causes the development of yellow colour.
Zex=negative control (MVA-BN infected cells).

[0047]FIG. 2: GUS activity after early and early/late expression

[0048]Cells were infected with MVA-BN and transfected with the appropriate
plasmids. After 24 hours cells were extracted and the GUS activity was
determined indirectly by measuring the extinction at 415 nm after an
enzymatic reaction, which causes the development of yellow colour.
Zex=negative control (MVA-BN infected cells). For that enzymatic
reaction, samples without AraC (early+late) expression had to be
incubated for 5 hours, the one with AraC (early expression) had to be
incubated over night in order to obtain a color reaction.

[0049]FIG. 3: GUS activity after expression by different promoters

[0050]Cells were infected with MVA-BN and transfected with the appropriate
plasmids. After 24 hours the cells were extracted and the GUS activity
was determined indirectly by measuring the extinction at 415 nm after an
enzymatic reaction, which causes the development of yellow colour.
Control=negative control (MVA-BN infected cells).

EXAMPLES

[0051]The following example(s) will further illustrate the present
invention. It will be well understood by a person skilled in the art that
the provided example(s) in no way may be interpreted in a way that limits
the applicability of the technology provided by the present invention to
this example(s).

Example 1

Analysis of Promoters to Express Coding Sequences in the MVA-BN Genome

1.1 Aim of the Experiment

[0052]It was the aim of this analysis to identify promoters that are
suitable to express coding sequences in the MVA genome, preferably coding
sequences that are heterologous to the natural MVA genome. Especially for
the insertion of two or more genes in a single insertion site it is
advantageous to use different promoters for expression of the single
genes in order to reduce the risk of recombination events, which could
result in deletion of one of the foreign genes. Furthermore it is
desirable to have promoters of different strength in order to have the
possibility to express the foreign genes inserted in recombinant MVA-BN
in variable amounts, depending on the strength of the promotor. 11
putative promoters were isolated in total. These putative promoter
sequences were cloned in a plasmid backbone (pBSKS+). In order to analyse
their potential activity, the promoters were fused to the GUS (E. coli
β-Glucuronidase) reporter gene. BHK (baby hamster kidney) cells were
infected with MVA-BN and transfected with the plasmids containing the
putative promoters fused to the GUS gene. If the promoter was functional,
GUS was expressed and could be quantified by an enzymatic reaction of
GUS. As positive control and as reference the well-characterized Vaccinia
virus promoter's p7.5 and Ps were fused to GUS and analysed in parallel.
By measurement of the GUS expression the putative promoters were screened
on activity, strength and early/late expression. The early/late
expression was checked by adding AraC (Arabinosid Cytosine) to the
culture media. The promoters, which are shown to be functional, namely
the Ps, p7.5, 7.5L and ATI promoter which were known in the prior art as
well as the newly identified promoter sequences that are naturally
involved in the expression control of the MVA ORF's, A42L, B9R, C7L, F6R,
I2R, J6R preferably can be to used for the expression of foreign genes in
recombinant MVA constructs (recMVA-BN).

[0054]5×105 BHK cells were seeded per transfection reaction in
a well of a 6-well-plate and maintained in DMEM/10% FCS over night at
37° C. and 5% CO2.

[0055]Infection/transfection Cells were infected with MVA-BN (moi 0.1) in
0.5 ml DMEM/10% FCS per well and incubated for 1 h at room temperature on
a shaker. Transfection was performed as described in the manufacturers
protocol. 2 μg plasmid were diluted in buffer EB (100 μl total
volume). After addition of 3.2 μl enhancer solution the solution was
mixed and incubated for 5 min. at room temperature. Then 10 μl
Effectene reagent was added, suspension was mixed and incubated for 10
min. at room temperature. The virus-suspension was removed from the cells
and 1.6 ml DMEM/10% FCS were added. 0.6 ml DMEM/10% FCS were added to the
DNA Effectene mixture and dropped on the cells while rotating the culture
plate. Cells were then incubated 7, 24 or 48 hours dependent on the
analysis. For the analysis of early/late expression AraC was added to the
medium during infection and transfection (40 μg/ml).

Harvesting of the Cells

[0056]Medium was removed from cells and 0.5 ml of Lysis buffer was added.
After shaking 15 min. at RT, cells were scraped in the Lysis buffer,
transferred to a 1.5 ml reaction tube and vortexed vigorously. Lysed
cells were centrifuged for 1 min. at 500 rcf and 4° C., the clear
supernatant was transferred to a fresh vial and stored at -20° C.
until use.

Determination of GUS Activity

[0057]10 μl of cell extract (=protein out of 2×104 cells)
was added to 1 ml prewarmed substrate solution (37° C.) and
incubated at 37° C. until a yellow colour was developed. Samples
were then placed on ice immediately and 0.4 ml stop solution was added.
The Extinction at 415 nm was determined and equated with the GUS activity
as extinction values between 0.05 and 2.0 are in a linear range. The
substrate solution was used as reference and a cell extract of MVA-BN
infected cells was used as negative control.

1.4 Experiments and Results

Experiment 1

Determination of Function of Putative Promoters

[0058]For the first experiment all plasmids, which contain a putative MVA
promoter or a well-characterized promoter fused to the GUS gene, were
analysed. Cells were infected with MVA-BN (moi 0.1) and transfected with
the corresponding plasmid. Cells were harvested after 48 hours, lysed and
GUS activity was determined. This experiment was performed in order to
determine, which promoters are functional. The results are shown in FIG.
1.

[0059]The negative control (extract from MVA-BN infected cells) clearly
showed no GUS activity (Zex; extinction 0.001). The well-characterized
strong synthetic Ps promoter was shown to be very efficient (Ps;
extinction 0.87) as there was a high amount of GUS detectable after 48
hours of expression. The also well-known naturally occurring Vaccinia
virus pr7.5 promoter did show also a quite high activity (p7.5;
extinction 0.41). Also the late portion of pr7.5 (7.5L; extinction 0.25)
shows a clearly detectable activity. The Cowpox ATI promoter clearly
showed to be very efficient for the expression of foreign genes by MVA-BN
(ATI; extinction 0.76). For the putative promoter regions of the MVA-BN
genome, it was shown that A42R (A42; extinction 0.48), 89R (B9;
extinction 0.06), C7L (C7; extinction 0.055), F6R (F6; extinction 0.208),
I2R (12; extinction 0.130) and J6R (J6; extinction 0.290) are functional
promoters. The promoters, which clearly showed to be active
(extinction>0.05) in the first preliminary experiment, were
characterized in more detail (Experiment 2).

Experiment 2

Characterization of Expression of Promoters

[0060]The promoters, which did show activity in experiment 1 were
characterized on their pattern of expression. For that purpose, the cells
infected with MVA-BN and transfected with the corresponding plasmid were
incubated with AraC. AraC inhibits the DNA replication, which is an
essential prerequisite for the late expression of genes during the MVA
replication cycle. In parallel the same experiment was performed without
addition of AraC. Infected and transfected cells were harvested after 24
hours and the GUS activity was determined in triplicate. FIG. 2 shows the
average extinction of each sample.

[0062]The promoters B9, C7 and J6 were shown to be mainly involved in the
late expression during the life cycle of MVA, as incubation with AraC
(+AraC), which inhibits late expression, results in an expression-level
of GUS comparable to that of the negative control (Zex). Although the C7L
promoter appeared to be rather weak several hints exist, that it plays an
important role during early expression.

[0063]The promoters A42R, I2R and F6R clearly show a very efficient early
expression. As for the determination of the early expression the samples
had to be incubated over night in order to get a detectable color
reaction. These results cannot be compared to the values of the
early+late expression (FIG. 2: -AraC) directly as they only were
incubated for 5 hours. The promoters, which did show early expression
were analysed again after 7 hours of expression and the results after 24
hours could be confirmed (data not shown).

1.5 Conclusion

[0064]It was clearly shown, that promoters of different strength could be
obtained and that there is now a spectrum of different promoters
available, which show different expression patterns dependent on the
incubation period and on the possibility of MVA-BN to replicate. If
early+late expression is preferred, promoters A42R, I2R and F6R are
preferably used. If the early expression should be avoided (e.g. for
foreign genes, which contain the stop signal TTTTTNT for early
expression), the promoters B9R, J6R or C7L are preferably used.

Example 2

Analysis of Minimal Promoter Elements Derived from SEQ ID NO: 1 to 4

[0065]In example 1 several sequences have been identified that are
particularly suitable to express foreign genes in the MVA genome. In
order to check whether shorter fragments fulfill the same purpose
additional experiments were carried out. Shorter fragments of SEQ ID NO 1
to 4 were isolated by PCR and cloned in a plasmid backbone (pBSKS+). In
total 6 putative minimal promoters were tested. In order to analyse their
potential activity, the promoters were fused to the GUS reporter gene.
BHK cells were infected with MVA-BN and transfected with the plasmids
containing the putative minimal promoters fused to the GUS gene. By
measurement of the GUS expression the putative promoters were screened on
activity and strength of expression (see example 1). As positive control
and as reference the well-characterized Vaccinia virus late promoter Ps
was fused to GUS and analysed in parallel. The minimal promoter elements
of about 30 bp can be used for the expression of foreign genes in
recombinant MVA constructs (recMVA-BN) without the risk of homologous
recombination between the homolgous sequences of the original and the
additionally cloned promoter.

2.1 Material and Method

[0066]If not indicated otherwise the materials and methods used in example
2 correspond to the methods in example 1. PCR was made according to
standard techniques.

2.2 Experiments and Results

Fusion of Promoters to GUS Gene by PCR

[0067]The PCR reactions resulted in the fusion of the following minimal
promoter sequences to the GUS gene:

[0068]All putative minimal promoters fused to the GUS gene were cloned in
pBSKS+ and sequenced.

Determination of Function of the Putative Minimal Promoters

[0069]In order to analyse functionality of the putative minimal promoter
elements the BHK cells were infected with MVA-BN (moi 1.0) and
transfected with the corresponding plasmid. Cells were harvested after 24
hours, lysed and GUS activity was determined (FIG. 3).

[0070]The negative control (extract from MVA-BN infected cells) clearly
showed no GUS activity (control; average extinction 0.00167). The
well-characterized strong synthetic Ps promoter was shown to be very
efficient (Ps; average extinction 2.05267) as there was a high amount of
GUS detectable after 24 hours of expression. For the putative promoter
minimal promoter elements of the MVA-BN genome, it was shown that all of
F6R short early, F6R short late, I2R short early, I2R short late, A42R
short late and J6R short late are functional promoters.

[0071]In total six functional minimal promoter elements were isolated. Two
are suitable for the weaker early transcription (I2R short early: average
extinction 0.06933; F6R short early: average extinction 0.189) and four
are suitable for late expression of different levels (F6R short late:
average extinction: 0.09833; I2R short late: average extinction 0.391;
J6R short late: average extinction: 0.80167 and A42R short late: average
extinction 2.07).

2.3 Conclusion

[0072]It was clearly shown, that promoters of different strength could be
isolated and that there is now a spectrum of different promoters
available. If early expression is preferred, the minimal promoter F6R
short early or I2R short early are preferably used. If the late
expression is preferred and early expression should be avoided (e.g. for
foreign genes, which contain the stop signal TTTTTNT for early
expression), the minimal promoter elements F6R short late, I2R short
late, J6R short late and A42R short late are preferably used.